Bacillus strains produce and secrete a large number of useful proteins and metabolites. Because of their GRAS (General Recognised As Safe) status, Bacillus hosts, e.g. belonging to the species Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis, are largely used in the production of important proteins for the food, feed and pharmaceutical industry.
A common problem in the application of Bacillus hosts in the expression of heterologous or the overexpression of homologous proteins of interest is related to the presence in said host cells of genes coding for several extracellular proteases which are co-expressed together with the proteins of interest. For those heterologous proteins of interest which are sensitive to protease degradation, the co-expression of homologous proteases in Bacillus leads to loss of yield, while the homologous Bacillus proteins of interest are contaminated by said co-expressed Bacillus proteases leading to purification problems.
The major extracellular protease in Bacillus hosts are extracellular neutral proteases (also known as bacillolysin EC 3.4.24.28) and alkaline proteases (also known as subtilisin EC 3.4.21.62). The problem caused by extracellular proteases in the production of useful proteins by Bacillus strains can be solved by using a Bacillus strain which is deficient in said extracellular neutral and alkaline proteases (see for example Kawamura et al (Journal of Bacteriology, (1984) p. 442-444, which describes a mutant strain of Bacillus subtilis which is deficient in the structural genes for extracellular neutral (nprE) and alkaline (aprA) proteases).
Other genes present in Bacillus strains have been shown to decrease the productivity of proteins of interest in said production hosts. U.S. Pat. No. 7,585,674 discloses that the productivity of a protein or polypeptide of interest in host cells, e.g. Bacillus cells, can be improved by deleting from the genome of said host cell specific genes participating in sporulation stage II, III, IV or V.
However there still remains a need for methods to increase expression of proteins in Bacillus hosts.